please give me some suggestions on preparating Yeast cells with PI stainning fo - buffer ,PI solution (Sep/12/2007 )

Hi, now i am going to analysis the cell cycle of yeast with PI stainning by Flow Cytometry .
now i have some problems:
first,which buffer is better for yeast cell, PBS or Tris-Hcl ?
second,how to use the PI,RNase,Tritonx-100,Pepsin ,Na citrate?

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S.cerevisiae or Pombe? Probably doesnt matter, I would suggest just using 1x PBS (ice cold) as a wash, fix 1x10^6 cells in 70% COLD Ethanol overnight, wash again several times in COLD PBS and then resuspend in 500ul - 1000ul of PI for 1 hour. You might get better results if you use a lyticase for the cell wall, but I don't think this is really neccessary.

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S.cerevisiae or Pombe? Probably doesnt matter, I would suggest just using 1x PBS (ice cold) as a wash, fix 1x10^6 cells in 70% COLD Ethanol overnight, wash again several times in COLD PBS and then resuspend in 500ul - 1000ul of PI for 1 hour. You might get better results if you use a lyticase for the cell wall, but I don't think this is really neccessary.
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Hi,jonathanjacobs:
thanks for your advice.
but now i still have a question
what does the PI solution should contain for S.cerevisiae ，PI,RNase,Tritonx-100,Na citrate?

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Well, to be honest, I'm not sure. We use a PI Solution that is pre-forumalted from BD Pharmacia in our lab. Here's the link to the product we use

http://www.bdbiosciences.com/external_file...211E_556463.pdf

it is 50mg/ml of PI in PBS i believe.

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OH,according to what you said,the concentration of PI is 25mg/ml per 1x10^6 cells,is it for Saccharomyces cerevisiae?

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